The idea that common pathogenic events exist at least for some autoimmune disorders is suggested by the significant number of patients displaying more than one autoimmune disease, and also by the strong and common linkage that some of these diseases show to specific MHC haplotypes. The experimental observation that the autoantigen is the leading moiety in autoimmunity and that a limited number of self-components are autoantigenic, suggests that these self-components share biological features which are relevant for self/non-self recognition by the immune system. One possibility is that triggering events by altering these features result in abnormal proteolysis. In certain individuals expressing a particular MHC specificity, the resulting abnormal peptides could be recognized by non-tolerized T cells and trigger an immune response
Type IV collagen (also referred to herein as collagen IV) networks scaffold the basement membranes, the laminar extracellular matrix structures often found between the cells and connective tissue. Six different type IV collagen α chains (α1-α6) exist, and three chains associate through the C terminal non-collagenous (NC1) domain to form a collagen IV molecule. In basement membranes, two type IV collagen molecules interact through their NC1 regions, yielding a hexameric globular quaternary structure (“hexamer”). Six disulfide bonds stabilize the native structure of each individual NC1 domain, and bonds generated by disulfide exchange between collagen IV molecules stabilize the “hexamer”. Bacterial collagenase digestion of basement membrane degrades the collagenous portion of collagen IV and releases the “hexamer”. Upon dissociation, this globular structure yields the individual NC1 domains as single polypeptides (“monomer”) or disulfide-related oligomers (dimers and higher molecular weight aggregates).
Recent data indicates that the information required to form a collagen IV “hexamer” resides in the covalent structure of the “monomer” as the individual NC1 domains select their partners and form “hexamers” without the assistance of other cellular factors. However the structural features mediating “monomer” association and the mechanism regulating the intermolecular disulfide bridging is presently unknown.
The chain composition of the collagen IV network varies among basement membranes and different collagen IV networks have been shown to exist. In the kidney, the glomerular basement membrane (GBM) results from assembly of two connected but independent collagen IV networks, one containing α1-α2(IV) and the other made of α3-α4-α5(IV). GBM plays a major role in plasma ultrafiltration since genetic and acquired diseases altering its collagen IV network impair renal function. In Alport syndrome, mutations in any of the α3, α4 or α5(IV) genes result in disruption of the corresponding collagen IV network and nephritis, whereas in Goodpasture (GP) disease an autoimmune response against the α3(IV)NC1 (also referred to as the GP antigen) cause linear deposits of autoantibodies along alveolar and glomerular BM, causing a rapidly progressive glomerulonephritis and often lung hemorrhage.
In GP disease, immunologically privileged epitopes buried in the GBM hexamer are exposed by an unknown pathogenic mechanism that engages the immune system in the deleterious production of antibodies. The human condition of this disorder and the exclusive involvement of the α3(IV)NC1 domain among six highly related domains, supported early comparative studies to identify biological features relevant in autoimmune pathogenesis. Accordingly, the human α3(IV)NC1 domain undergoes unique phosphorylation at Ser9 by type A protein kinases (cPKA) and structural diversification by alternative exon splicing generating multiple related products (GPΔIII, GPΔIII/IV/V and GPΔV).
The data presented herein indicate that the human α3(IV)NC1 domain exists as multiple phosphorylation-dependent conformational isoforms (conformers) that are stabilized by disulfide bonds. Furthermore our data indicate that phosphorylation of Ser9 induces conformational diversification of the α3(IV)NC1 domain, whereas the alternative products contain divergent C terminal ends that specifically induce cPKA phosphorylation of Ser9 in the primary product, suggesting that in humans the levels of expression of alternatively spliced products by regulating Ser9 phosphorylation control the conformational diversification process of the α3(IV)NC1 domain. All of the above suggests that Ser9 phosphorylation, alternative exon splicing and pathogenesis are related phenomenon.
The data presented herein further identify GPBP and GPBPΔ26 as two alternatively spliced isoforms of a novel non-conventional protein kinase that binds to the N terminal region of the human Δ3(IV)NC1 and phosphorylates Ser9. GPBP is a more active variant whose expression is highly restricted to histological structures targeted by common autoimmune responses including human alveolar and glomerular basement membranes. Each GPBP isoform likely represents a different strategy to perform the same function as we have found that for a particular tissue individuals expressing higher levels of GPBP express very little GPBPΔ26 and vice versa. An augmented expression of GPBP with respect to GPBPΔ26 has been associated with several autoimmune conditions including GP patients, cutaneous lupus erythematosus, pemphigus, pemphigoid and lichen planus, suggesting that GPBP expression and autoinmnune pathogenesis are related processes. Our data herein (Example 5) further indicate that phosphorylation activates the α3(IV)NC1 domain for aggregation, a process that is catalyzed at least in part by GPBP and which comprises conformational isomerization reactions and disulfide-bond exchange.
Furthermore we show here that in GP kidneys, a relative increased in the level of expression of GPΔIII and GPBP co-exist with assembled “aberrant” conformers of the α3(IV)NC1 domain that conduct the autoimmune response, suggesting this human disease represents the legitimate response of the immune system against misfolded autoantigen which results from a coordinated increase in the expression of GPBP and GPΔIII.
Finally, we disclose that myelin basic protein (MBP), a known human autoantigen implicated in multiple sclerosis, contains a structurally related site (Ser8) for cPKA and GPBP whose phosphorylation regulates conformation and is under the control of a related alternative splicing mechanism when cPKA is phosphorylating enzyme, suggesting that phosphorylation-dependent conformation is the biological condition that renders self-components potentially immunogenic.
Based on all of the above, there exists a need in the art for methods and reagents to identify drug candidates to modify GPBP activity to treat autoimmune disorders.